The main hindrance to develop antiviral therapies and effective vaccines for HIV-1 is the high variability of the virus. Recombination is a major mechanism that is responsible for the rapid diversification of HIV-1 population. Recombination between different subtypes of HIV-1 generates intersubtype recombinants. These intersubtype recombinants can also recombine with HIV-1 of the same or different subtypes or with other recombinants to generate more complex recombinants. Intersubtype recombinants emerge in almost every region of the world where more than one HIV-1 subtype is present. Currently, 49 HIV-1 circulating recombinant forms (CRFs) and a large number of unique recombinant forms (URFs) have been identified. These CRFs and URFs accounted for approximately 20% of global infections and they continue to play an increasingly important role in shaping the AIDS pandemic. iVly current research focuses on the mechanisms and pathogenomics of HIV-1 replication and intersubtype recombination. In this application, we continue our study on understanding the molecular mechanisms for generating HIV-1 intersubtype recombinants with biological advantages. Our long-term goal is to elucidate the elements in the viral genome that affect replication fitness of a HIV-1 recombinant. These viral elements represent new potential targets for blocking or enhancing recombination that can affect the continuous replication of HIV-1 in the host. The objectives of this application are to characterize the molecular structures and the replication fitness of HIV-1 intersubtype recombinants generated in vivo. The central hypothesis is that newly generated HIV-1 intersubtype recombinants have an array of replication fitness and there is a finite window for HIV-1 replication fitness allowing the recombinants to continue to replicate in the host. The Specific Aims are to 1) reveal the diversity of HIV-1 intersubtype recombinants in vivo, 2) characterize the replication fitness of HIV-1 intersubtype recombinants and 3) define the selection pressure for generating HIV-1 intersubtype recombinants with biological advantages. We expect our work will enhance our knowledge on the molecular mechanisms that generate HIV-1 variations and new HIV-1 recombinant strains.